Disruption of target DNA binding in Mu DNA transposition by alteration of position 99 in the Mu B protein.
Target DNA binding by the Mu B protein is an important step in phage Mu transposition; however, the region of Mu B involved in target binding and the mechanism of the interaction are unknown. Previous studies have demonstrated that modification of Mu B with the sulfhydryl-specific reagent N-ethylmaleimide can selectively inhibit target DNA binding. We now show that individual mutation of the three cysteines in Mu B to serine results in proteins which are active in intermolecular strand transfer, but demonstrate variable levels of N-ethylmaleimide resistance. The data indicate that cysteine 99 is the primary site of modification affecting target DNA binding, with a minor contribution resulting from the derivatization of cysteine 129. These findings are confirmed by the construction of Mu B mutants containing a bulky side-chain at the individual cysteine to mimic the N-ethylmaleimide modified protein. The C99Y protein shows a complete loss in target-dependent strand transfer activity under standard reaction conditions and C129Y displays partial activity. The effect of the tyrosine substitutions is specific for target interaction as both mutants show wild-type activity in their ability to stimulate the Mu transposase to perform donor cleavage and intramolecular strand transfer. Finally, a target dissociation assay has shown that the C99Y-DNA complex generated in the presence of ATP-gamma-S has a drastically reduced half-life as previously found for N-ethylmaleimide treated wild-type Mu B. Modification of cysteine 99 is proposed to block target DNA binding by causing steric interference near the DNA binding pocket.
Millner
A
A
Department of Biochemistry, University of Western Ontario, London, Canada.
Chaconas
G
G
D019943:Amino Acid Substitution; D010583:Bacteriophage mu / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D019895:Transposases / Q000378:metabolism*; D014764:Viral Proteins
D004268:DNA-Binding Proteins; C050938:Mu B protein, bacteriophage; D014764:Viral Proteins; D019895:Transposases; D003545:Cysteine; D005033:Ethylmaleimide